The branched chain ketoacid dehydrogenase (BCKD) is the rate-limiting step in the catabolism of leucine, isoleucine and valine in many tissues. I have recently demonstrated that this enzyme can be inactivated by phosphorylation. The elucidation of this control mechanism for BCKD is essential in understanding branched chain amino acid catabolism. This pathway is involved in the physiological regulation of glucose production and nitrogen balance during catabolic states such as starvation and trauma. There are four basic goals in this proposal: (1) The purification of BCKD and analysis of the phosphorylation stoichiometry. Since relatively few phosphorylated proteins have been studied in detail, these experiments will add to our understanding of the specificity of phosphorylation sites and the role of multiple phosphorylation sites in proteins. (2) Purification of the BCKD kinase and characterization of its properties. This enzyme has an apparently unique specificity since other known kinases do not phosphorylate BCKD. Attempts to purify the phosphatase are also described. To carry out the proposed studies, BCKD and its kinase will be purified from pig kidney. (3) A study of the effects of potential modifiers of BCKD and BCKD kinase. These studies will also quantitatively distinguish the regulatory importance of phosphorylation vs. feedback inhibition by end products as a mechanism for the control of BCKD activity in the mitochondrial environment. In addition the possible role of mitochondrial transport of branched chain ketoacid as a limiting step in amino acid metabolism will be examined. (4) Measurement of the phosphorylation state of BCKD in vivo. Methods are devised for isolation of BCKD without altering its phosphorylation state. The regulatory properties responsible for the altered activity will then be analyzed. It is hoped that these studies will lead to an elucidation of the physiological regulation of branched chain amino acid catabolism.